1. Field of the Invention
This invention relates to the isolation and characterization of novel Classical Swine Fever Virus (CSFV) virulence determinants associated with E1 glycoprotein function and E2 glycoprotein function and utilization of these novel virulence determinants to design live attenuated CSF vaccines.
2. Description of the Relevant Art
Classical swine fever (CSF) is a highly contagious and often fatal disease of swine, which is characterized by fever and hemorrhages and can present with either an acute or a chronic course (van Oirschot, J. T. 1986. In: Diseases of Swine, 6th edition, Leman et al., eds., Iowa State University Press, Ames, Iowa, page 289). The causative agent is classical swine fever virus (CSFV). Infection with highly virulent CSFV strains generally leads to death in infected animals, whereas isolates of moderate to low virulence induce a prolonged chronic disease. The genetic basis of CSFV virulence and host range is poorly understood (van Oirschot, supra).
CSFV is enzootic in all continents, and actively circulating in South and Central America, southern Mexico, and the Caribbean. Disease outbreaks occur intermittently in Europe where control programs of the disease (Westergaard et al., 1998. In: Vaccines in Agriculture: Immunological Applications To Animal Health and Production, Wood et al., eds., CSIRO, East Melbourne, Australia, pages 13-20) include quarantine and eradication of infected herds, resulting in the elimination of a large number of animals, including noninfected animals, thus engendering significant economic losses. Vaccination, quarantine and testing protocols offer an alternative to these measures and may become the only means to control and eradicate an outbreak of CSF, reducing the economic impact that results from the heretofore elimination of such a vast number of pigs.
Safe and effective CSFV vaccines that prevent clinical symptoms of the disease and virus spreading, including during the early post-vaccination period, have been used around the world (Aynaud, J. M. 1988. In: Classical Swine Fever and Related Viral Infections, B. Liess, ed., Nijhoff, Boston, Mass., pages 165-176; Biront and Leunen. 1988. ibid., pages 181-197). Among available CSFV vaccines, live attenuated vaccines, such as C strain, GPE-strain, Thiversal strain, and PAV-250, confer effective and long lasting immunity against CSF (Biront and Leunen, ibid.). In general, these vaccines have been obtained after serial passages of CSFV isolates in tissue culture or rabbits; however, the genetic bases of the attenuation in the above cases are unknown. Additionally, it is not currently possible to distinguish, serologically, between animals vaccinated with live attenuated vaccines and animals infected with wild-type virus.
Development of infectious CSFV cDNA clones has enabled genetic approaches for defining mechanisms of viral replication and pathogenesis. Infectious clones of the attenuated CSFV C-strain and the pathogenic Alfort/Tübingen strain have been constructed, enabling identification of Ems and Npro as virulence factors in swine and the role of different Ems mutations in virus attenuation (Meyers et al. 1996, supra, Moormann et al. 1996. J. Virol. 70: 763-770, Ruggli et al. 1996. J. Virol. 70:3478-3487, Mayer et al. 2004. Vaccine 22: 317-328). CSFV infectious clones have been used to identify viral proteins or protein domains functioning in viral replication and virulence, and to engineer attenuated marker CSF live attenuated vaccines (Meyers et al. 1999, supra, Moser et al. 2001. Virus Genes 23: 63-68, Tratschin et al. 1998. J. Virol. 72: 7681-7684, van Gennip et al., supra).
CSFV is a member of the Pestivirus genus of the Flaviviridae family (Francki et al. 1991. Archives of Virol. Suppl. 2:223). CSFV is a small enveloped virus with a single-stranded, 12.5 kb RNA genome of positive polarity which contains a long open reading frame encoding a 3000 amino acid polyprotein with a gene order: NH2-Npro-C-Ems-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. The polyprotein gives rise to 11 to 12 final cleavage products by co- and posttranslational processing involving cellular and viral proteases (Rice, C. M. 1996. In: Fundamental Virology, Third edition, Fields et al., eds., Lippincott Raven, Philadelphia, Pa., pages 931-959). Protein C and the glycoproteins Ems, E1 and E2 represent structural components of the virion (Thiel et al. 1991. J. Virol. 65: 4705-4712). E1 and E2 are anchored to the envelope by their carboxy termini, with Ems loosely associated with the envelope. Ems and E2 are present as homodimers linked by disulfide bridges on the surface of CSFV virions; E2 is also found dimerized with E1 (Weiland et al. 1990. J. Virol. 64: 3563-3569; Weiland et al. 1999. J. Gen. Virol. 80 (Pt 5): 1157-1165). Although Ems and E2 have been shown to function in viral pathogenesis and the induction of a protective immune response, respectively (Hulst et al. 1997. J. Gen Virol. 78 (Pt 11): 2779-2787; Hulst et al. 1998. J. Virol. 72: 151-157; Meyers et al. 1999. J. Virol. 73: 10224-10235; Meyers et al. 1996. J. Virol. 70: 1588-1595; van Gennip et al. 2000. Vaccine 19: 447-459; Weiland et al. 1990, supra), the function of E1 remains unknown. The genetic basis of CSFV virulence and host range remains poorly understood (van Oirschot, supra).
Candidate CSFV subunit marker vaccines have been developed using recombinant E2 envelope protein (Van Zijl et al. 1991. Vaccine 17: 433-440; Hooft van Iddkinge et al. 1996. Vaccine 14: 6-12; Hulst et al. 1993. J. Virol. 67: 5435-5442; Van Rijn et al. 1996. J. Gen. Virol. 77: 2737-2745; Van Rijn et al. 1998. Vaccine 17: 433-440). E2, the major structural protein, induces neutralizing antibodies and protective immunity in infected and vaccinated pigs (Wensvoort et al. 1989. Vet. Microbiol. 21: 9-20; Van Zijl et al., supra; Hulst et al., 1993, supra; Rumenapf et al. 1991. J. Virol. 65: 589-597; Van Rijn et al., supra). Different E2 protein domains have been described as targets for neutralizing monoclonal antibodies (Wensvoort et al., supra), but E2 subunit vaccines have not been found to be as efficacious as traditional live attenuated vaccines, particularly when animals are challenged shortly after vaccination (Hulst et al., 1993, supra; Van Rijn et al., supra; Risatti et al. 2003. J. Clin. Microbiol. 41: 500-505). The failure to induce rapid and efficient protective immunity precludes the use of subunit vaccines as an emergency control measure during a CSFV outbreak. DNA vaccines encoding E2, when expressed, also induced protection in pigs; however, again, rapid elicitation of protection was not proven (Andrew et al. 2000. Vaccine 17: 1932-1938; Yu et al. 2001. Vaccine 19: 1520-1525).
Ems has been shown to function in viral virulence (Meyers et al. 1999, supra). Depending on the extent of the engineered mutation, specific Alfort/187 Ems mutants exhibited different degrees of attenuation when inoculated in swine, indicating a role for Ems in viral pathogenesis (Meyers et al. 1999, supra). Additionally, attempts to mutate and inactivate Ems in the attenuated C-strain resulted in a virus atypically cytopathic for cell cultures (Hulst et al. 1998, supra, Widjojoatmodjo et al. 2000. J. Virol. 74: 2973-2980).
CSF infectious clones have also been used to examine the function of other viral proteins and genomic regions. The Npro gene of the Alfort/187 strain was replaced with the ubiquitin gene as a marker. In vitro replication kinetics of the recombinant and parental strains were indistinguishable, indicating that Npro is non-essential for virus growth in vitro (Tratschin et al., supra). Similarly, insertion of 44 bases within the 5′ non-translated region of the Alfort/187 genome also failed to affect in vitro replication kinetics (Moser et al., supra). Thus, CSFV infectious clones hold great promise for identifying viral proteins or protein domains functioning in viral virulence and host range.
Several live vector vaccines have been developed, using vaccinia, pseudorabies and adenoviruses (Van Zijl et al., supra; Rumenapf et al., supra; Konig et al. 1995. J. Virol. 69: 6479-6486; Peeters et al. 1997. J. Gen. Virol. 78: 3311-3331; Hammond et al. 2000. Vaccine 18: 1040-1050); however, limited potency, the possibility of antibodies specific for the vectors, and the unwanted introduction of recombinant vaccinia or adenovirus into the swine population have discouraged the use of this approach for controlling CSFV.
Recently, infectious clone technology has enabled antigenic modification of attenuated CSFV strains for use as experimental marker live attenuated CSF vaccines. Infectious clones of the attenuated C-strain were used to replace the antigenic region of E2 and/or the complete Ems gene with analogous sequences from Bovine Viral Diarrhea Virus (BVDV). Preliminary data indicated that both chimeric viruses were able to induce protection in pigs at one week after vaccination. Significantly, chimera-induced anti-CSFV antibody responses could be discriminated from those produced by parental virus (van Gennip et al., supra; Risatti et al., supra). Additionally, this approach, based on the replacement of homologous regions of the antigenically related Pestivirus (BVDV) in the genome of the attenuated vaccine C-strain, precludes the introduction of positive/negative markers in specific areas of the genome due to the lack of knowledge of the virulence host range determinant in CSFV. Recently, encouraging results have been obtained with a C-strain Ems deletion mutant complemented in trans with an Ems expressing cell line, resulting in a non-replicative live attenuated vaccine (Widjojoatmodjo et al., supra). Further experiments will be conducted to validate elicitation of rapid protection, as is provided by the replicative parental C-strain.
Thus, knowing the determinants of virulence would allow the introduction of precise modification at particular sites of the genome that will result in the attenuation of the virus. If antigenic markers are genetically engineered at those sites, such attenuate viruses can be applied as marker vaccines to the control of a CSF outbreak, allowing the identification and eradication of infected animals/herds, thereby preventing the elimination of uninfected animals.